Polyether antibiotic mi215-nf3 substance, production process thereof, and agent for control of chicken coccidiosis

ABSTRACT

In this invention, a new microbial strain which is a strain of Actinomycetes and belongs to the genus Actinomadura, namely Actinomadura sp. MI215-NF3 strain is cultured, and MI215-NF3 substance, a novel antibiotic classifiable as a polyether antibiotic, is recovered from the resultant culture. MI215-NF3 substance and its salts obtained according to this invention are useful for therapeutic treatment of chicken coccidiosis and also have useful antibacterial activities against certain species of bacteria.

TECHNICAL FIELD

This invention relates to a novel antibiotic substance MI215-NF3 andsalts thereof and also relates to a process for production of theMI215-NF3 substance or salts thereof. This invention further relates toagent for control of chicken coccidiosis, which contains the MI215-NF3substance or a salt thereof as an active ingredient. Furthermore, thepresent invention also pertains to a new microorganism, Actinomadura sp.MI215-NF3 strain which has characteristic capable of producing MI215-NF3substance.

PRIOR ART

Various antibiotics of polyether type are known to have antibacterialactivities. Monensin (see Japanese Patent Publication No. 113/70) andsalinomycin (see "The Journal of Antibiotics", 27, 814-821), whichbelong to the polyether antibiotics, are used as agents for control ofchicken coccidiosis.

Coccidiosis provides a serious problem in the poultry farming. There hashence been a continued desire for the discovery or development of a newcompound which can exhibit superior anticoccidial activity andproperties to the compounds previously known or used to date.Investigations are now made to achieve this end.

DISCLOSURE OF THE INVENTION

With a view toward discovering useful novel antibiotics, we, the presentinventors, have therefore proceeded with investigations. As a result, wehave succeeded in isolating a new strain belonging to the genusActinomadura. We have also found that this new strain can produce anovel polyether antibiotic. This antibiotic is now named MI215-NF3substance. Further, it has also been uncovered that this MI215-NF3substance and its salts (carboxylates) exhibit an activity to controlchicken coccidiosis. The present inventors have proceeded with theinvestigation further, to determine the chemical structure of MI215-NF3substance, whereby MI215-NF3 substance has been confirmed to be a novelcompound. In addition, it has also been found that MI215-NF3 substancecan be represented by the following formula (I): ##STR1## wherein R is ahydrogen atom.

Accordingly, this invention provides the substance which is obtained byculturing a microorganism belonging to the genus Actinomadura and whichhas been named as MI215-NF3 substance by the present inventors, as wellas salts thereof. This invention also provides a process for productionthereof, and their use as an agent for control of chicken coccidioses.

In a first aspect of the present invention, therefore, there areprovided MI215-NF3 substance having the above formula (I) and a saltthereof.

Although MI215-NF3 substance of the formula (I) is in the form of a freecarboxylic acid where R is a hydrogen atom in the formula (I), R in theformula (I)generally can be a metal or ammonium group. Therefore, thepresent invention also embraces salts of MI215-NF3 substance therein.These salts can be in the form of an alkali metal salt where R is analkali metal atom in the formula (I), for example, sodium salt,potassium salt and lithium salt, or in the form of an alkaline earthmetal salt where R is an alkaline earth metal atom in the formula (I),for example, calcium salt and magnesium salt. They can also be salts ofother metals, for example, aluminum salt and iron salt. The ammoniumsalt can also be included.

Physicochemical properties of MI215-NF3 substance according to thepresent invention are as follows.

The physicochemical properties of the sodium salt of MI215-NF3 substancewill be described hereinafter.

(1) Appearance

Colorless plate crystals.

(2) Elemental analysis

    ______________________________________                                               Found Calculated (for C.sub.37 H.sub.63 O.sub.11 Na)                   ______________________________________                                        C        62.47%  62.87%                                                       H         8.96%   8.98%                                                       O        24.60%  24.90%                                                       Na        3.53%   3.25%                                                       ______________________________________                                    

(3) Mass spectrometry (SIMS)

m/z 707(M+Na)

(4) Melting point

217-218° C.

(5) Specific optical rotation [a]²⁵ _(D=+) 30.6° (c1.0, chloroform)

(6) Infrared absorption specturm (KBr pellet method)

As be shown in FIG. 1 of the accompanying drawings.

(7) Proton nuclear magnetic resonance spectrum

A proton NMR spectrum as measured in deuterochloroform at 400 MHz androom temperature is as shown in FIG. 2 of the accompanying drawings.

(8) Solubility

Soluble in benzene, chloroform, acetone, ethyl acetate, ethanol andmethanol, but insoluble in water.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is an infrared absorption spectrum of the sodium salt ofMI215-NF3 substance as measured in accordance with the KBr pelletmethod.

FIG. 2 is a proton nuclear magnetic resonance spectrum of the sodiumsalt of MI215-NF3 substance as measured in deutero-chloroform at 400 MHzand room temperature.

Biological properties of MI215-NF3 substance according to the presentinvention are as follows.

The biological properties of the sodium salt of MI215-NF3 substance willbe described hereinafter.

(1) Antibacterial spectrum

The antibacterial spectrum of the sodium salt of MI215-NF3 substanceagainst various microorganisms was measured on an agar medium in amanner known per se in the art, using the serial dilution method. Theresults obtained are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                                             Minimum inhibitory                                                            concentration (MIC)                                      Microorganism tested (μg/ml)                                               ______________________________________                                        Staphylococcus aureus FDA 209P                                                                     <0.78                                                    Staphylococcus aureus Smith                                                                        1.56                                                     Micrococcus luteus FDA 16                                                                          1.56                                                     Micrococcus luteus IFO 3333                                                                        3.12                                                     Micrococcus luteus PCI 1001                                                                        <0.78                                                    Bacillus anthracis   0.78                                                     Bacillus subtilis NRRL B-558                                                                       1.56                                                     Bacillus subtilis PCI 219                                                                          1.56                                                     Bacillus cereus ATCC 10702                                                                         <0.78                                                    Corynebacterium bovis 1810                                                                         <0.78                                                    Escherichia coli NIHJ                                                                              >100                                                     Escherichia coli K-12                                                                              >100                                                     Escherichia coli K-12 ML 1629                                                                      >100                                                     Shigella dysenteriae JS 11910                                                                      25                                                       Salmonella typhi T-63                                                                              >100                                                     Proteus vulgaris OX 19                                                                             >100                                                     Serratia marcescens  100                                                      Pseudomonas aeruginosa A3                                                                          >50                                                      Klebsiella pneumoniae PCI 602                                                                      100                                                      Micobacterium smegmatis ATCC 607                                                                   12.5                                                     Staphylococcus aureus MS 8710                                                                      1.56                                                     Staphylococcus aureus MS 9610                                                                      <0.78                                                    Escherichia coli BE 1121                                                                           3.12                                                     Escherichia coli BE 1186                                                                           3.12                                                     Escherichia coli BEM 11                                                                            <100                                                     ______________________________________                                    

(2) Toxicity

When acute toxicity of sodium salt of MI215-NF3 substance was estimatedusing mice, the MI215-NF3 substance was suspended in physiologicalsaline containing 10% of dimethylsulfoxide. The suspension was injectedintraperitoneally, and the mice were observed for 14 days. As a result,the LD₅₀ value of the MI215-NF3 substance was then estimated to be 56mg/kg.

(3) Anticoccidial activity

The activity of MI215-NF3 substance for controlling chicken coccidiosiswas tested as summarized below.

1. Newly-hatched chicken (male) were preliminarily raised for 1 week,and two-week-aged chicken were used in the test.

2. The body weights of the chicken were individually measured at thebeginning of the test. To make up each test group, ten chicken wereselected and alloted in each group so as to minimize variations amongthe test groups.

3. Chicken were orally inoculated with 5×10⁴ oocysts of Eimeria tenellaper chick through a stomach probe tube.

4. After the oocyst inoculation, each chick was allowed to take adlibitum a feed which had been prepared by mixing a predetermined amountof MI215-NF3 substance (sodium salt) with a special feed containing noanticoccidial agent. Incidentally, the chicken were also allowed todrink water ad libitum.

5. Up to the 7th day after the oocyst inoculation, the number of oocystsin feces from each chick and the state of the feces were observedeveryday.

6. Each chick was anatomized on the 7th day after the infection.Anatomical procedures were as follows.

1) The body weight of each chick was measured.

2) Each chick was sacrificed by dislocation of neck cervical fracture.

3) Each chick was subjected to celiotomy and the cecum was enucleated.The cecum was visually inspected for any lesions on both outer and innerwalls of cecum.

4) The cecum thus inspected was individually disintegrated in ahomogenizer, whereby a cecum suspension was prepared. The number ofoocysts in the suspension was counted.

5) Based on the results of the above inspections, the value of ACI(Anticoccidial Index) was determined in accordance with the evaluationmethod proposed by Merck & Co., Inc., U.S.A., namely, by the followingequation:

    ACI=(rate (%) of relative body weight gain+survival rate (%) of chick)-(lesion scores+oocyst scores)

Upon determination of the above ACI value, the "rate of weight gain" ofthe chicken in each group of chicken during the test period forestimation of the drug efficacy and the "rate of relative body weightgain" used for the calculation of ACI were evaluated in accordance withthe following equations:

    ______________________________________                                        Rate (%) of body weight gain = [(mean body weight of                          chicken at the end of the test - mean body                                    weight of the chicken at the beginning of                                     the test) ÷ mean body weight of the chicken                               at the beginning of the test] × 100.                                    Rate (%) of relative body weight gain =                                       [(rate of body weight gain of the treated group                               or an infected but non-treated group) ÷ rate                              of body weight gain of a non-infected and non-                                treated group] × 100.                                                   ______________________________________                                    

On the other hand, the term "lesion scores" means a value which isobtained by evaluating, in terms of indexes, the results of visualinspection of lesions on an enucleated cecum sample. The term "oocystscores" means a value which is obtained by evaluating, in terms ofindexes, the number of oocysts actually counted. Details of theprocedure for the determination of ACI values are described, forexample, in the literature compiled by Kiyoshi Tsunoda, "ChickenCoccidiosis", pp 95-101, Chikusan Shuppan Sha (May, 1983).

The results obtained are summarized in Table 2.

                                      TABLE 2                                     __________________________________________________________________________                    Test results                                                  Test Plots      Dosage                                                                            Rate (%) of relative                                                                    Survival                                                                           Lesion                                                                            Oocyst                                 Test groups     (ppm)                                                                             body weight gain                                                                        rate (%)                                                                           scores                                                                            scores                                                                            ACI                                __________________________________________________________________________      Non-infected control group                                                                   0  100       100  0   0   (200)                                Group treated with                                                                          80  88.67     100  4   10  174.7                                Na salt of MI215-NF3                                                          Group treated with                                                                          100 99.39     100  10.0                                                                              1   188.4                                Na salt of MI215-NF3                                                          Group treated with                                                                          80  90.20     100  2   5   183.2                                Ca salt of MI215-NF3                                                          Group treated with                                                                          80  90.16     100  0   1   189.2                                Al salt of MI215-NF3                                                          Group treated with                                                                          100 96.13     100  32.0                                                                              20  144.1                                Monensin                                                                      Group treated with                                                                          50  71.13     100  34.0                                                                              10  127.1                                Salinomycin                                                                   Infected control group                                                                      0   89.63     100  40.0                                                                              30  119.6                              __________________________________________________________________________

As is apparent from the foregoing results, MI215-NF3 substance and itssalts are practically utilizable as an agent for controlling ortherapeutically treating chicken or fowl coccidiosis.

Further, in a second aspect of the present invention, there is alsoprovided a process for the production of the antibiotic, MI215-NF3substance or a salt thereof, characterized in that the process comprisesculturing a microorganism, which belongs to the genus Actinomadura andcan produce MI215-NF3 substance of the formula (I), and recoveringMI215-NF3 substance or a salt thereof from the culture of themicroorganism.

Microbiological properties of the MI215-NF3-substance-producingmicroorganism useful in the process of the present invention are asfollows:

Any MI215-NF3-substance-producing microorganism can be used in thepresent invention irrespective of its species, as far as it has thecapability to produce said antibiotic having the physicochemicalproperties and biological properties described above. TheMI215-NF3-substance-producing microorganism to be used in this inventioncan therefore be selected from a wide variety of microorganisms. Amongsuch microorganisms, one specific preferred example of theMI215-NF3-substance-producing microorganisms is a strain ofActinomycetes to which the strain designation, MI215-NF3, has beenallotted and which was isolated by the present inventors from a soilsample collected in Bunkyo-ku, Tokyo.

The microbiological characteristics of MI215-NF3 strain are describedbelow.

1. Morphology

In microscopic observations, aerial hyphae extend from branchedsubstrate mycelia. Substrate mycelia do not split. Spore chains areformed on aerial hyphae and are of a hooked or spiral shape. Maturedspore chains generally consist of 7-15 spores. Spores are of an ovoidalshape (with about 0.7×1.0-1.2 micrometers in sizes), and the surface ofspore is warty. Neither whirls nor sporangia are observed.

2. Conditions of Growth on Various Media

The standards given in the brackets [] for the correspondingdescriptions of colors are same as those given the "Color HarmonyManual" of Container Corporation of America.

(1) Sucrose-nitrate-agar medium (cultured at 30° C.)

On the growth of a colorless to pale orange color [3gc, Lt Tan], whitecolored aerial hyphae are formed thinnly. No soluble pigment isobserved.

(2) Glucose-asparagin medium (cultured at 30° C.)

On the growth of a pale yellow brown color [3ic, Lt Amber to 31e,Cinnamon] to light brown color [4ng, Lt Brown], white colored aerialhyphae are formal only in a small quantity. Soluble pigment is producedwith a somewhat yellowish tinge.

(3) Glycerin-asparagin-agar medium (ISP-Medium 5, cultured at 30° C.)

On the growth of a pale yellow to pale yellow brown color [21e, Mustardto 31e, Cinnamon], aerial hyphae of white to gray white[2dc Natural]color are formed thinnly. Soluble pigment exhibits a slight yellow browntinge from about the 21st day of incubation.

(4) Starch-inorganic salts-agar medium (ISP-Medium 4, cultured at 30°C.).

On the growth of a pale yellow to pale yellow brown [3ng, Yellow Maple]to yellow brown [3pi, Golden Brown]color, aerial hyphae of pink white[5ba Shell Pink] to gray white [2dc, Natural] color are formed. Solublepigment is produced with a slight yellow tinge.

(5) Tyrosin-agar medium (ISP-Medium 7, cultured at 30° C.)

On the growth of a pale yellow to yellow brown [3ng, Yellow Maple] tograyish yellow to yellow [3ni, Clove Brown] color, aerial hyphae ofyellowish gray [1dc, Putty-lec, Lt Citron Gray] color are formed.Soluble pigment is produced with a slight yellow tinge.

(6) Nutrient-agar medium (cultured at 30° C.)

On the growth of a pale yellow brown [3ic, Lt amber]to grayish yellowbrown [3ni, Clove Brown]color, white colored aerial hyphae are formedpartly. Soluble pigment is produced with an extremely slight yellowtinge.

(7) Yeast-malt-agar medium (ISP-Medium 2, cultured at 30° C.).

On the growth of a pale yellow brown [21e, Mustard] to grayish yellowbrown [2nl, Covert Brown], white to brown white colored aerial hyphaeare formed. Soluble pigment is produced with an extremely slight yellowtinge.

(8) Oatmeal-agar medium (ISP-Medium 3, cultured at 30° C.)

On the growth of a pale orange to pale brown [41e, Maple] to yellowbrown [3ng, Yellow Maple] color, aerial hyphae of white to pink white[5ba, Shell pink -7ba, Pink Tint] color are formed. Soluble pigment isproduced with a slight yellow tinge.

(9) Glycerin-nitrate-agar medium (cultured at 30° C. )

On the growth of a colorless to pale yellow to pale yellow brown [31e,Cinnamon] color, aerial hyphae of white to pink white [5ba, Shell Pink]color are formed. Soluble pigment is produced with a slight yellowtinge.

(10) Starch-agar medium (cultured at 30° C.)

On the growth of a pale yellow brown [21e, Mustard] to grayish yellowbrown [3ni, Clove Brown] color, white colored aerial hyphae are formedin a small quantity. No soluble pigment is observed.

(11) Calcium malate-agar medium (cultured at 30° C.)

On the growth of a colorless to pale yellow color, aerial hyphae ofwhite to pink white [5ba, Shell Pink ] are formed. Soluble pigment isnot observed.

(12) Cellulose (synthetic liquid added with pieces of filter paper)

The growth is colorless. White colored aerial hyphae are formed in asmall quantity. No soluble pigment is observed.

(13) Gelatin stab culture

On a plain gelatin medium (cultured at 20° C.), the growth is colorlessor shows pale yellow color. Neither aerial hyphae nor soluble pigmentare formed. On a glucosepeptone-gelatin medium (cultured at 27° C.), thegrowth is of a pale yellow color, and neither aerial hyphae nor solublepigment are observed.

(14) Skimmed milk (cultured at 37° C.)

The growth is of a pale yellow to pale yellow brown color. No aerialhyphae are formed. Soluble pigment is produced with a slightly browntinge from about the 18th day of the incubation.

3. Physiological properties (1) Temperature range for the growing

Using starch-inorganic salts-agar medium (ISP-Medium 4), tests forincubation of MI215-NF3 strain were conducted at 20° C., 24° C., 27° C.,30° C., 37° C. and 50° C., respectively. can grow at all the testedtemperatures except 50° C. It appears that optimal growth temperature isaround 30° C.

(2) Liquefaction of gelatin (15% plain gelatin medium, cultured at 20°C.; glucose-peptone-gelatin medium, cultured at 20° C.)

In the plain gelatin medium, liquefaction of gelatin started from about11th day after the incubation but is rather weak. In theglucose-peptone-gelatin medium, Liquefaction of gelatin is observed fromabout 5th day after the incubation and the grade of liquefaction ismedium to weak.

(3) Hydrolysis of starch (starch-inorganic salts-agar medium, ISP-Medium4 and starch-agar medium, all cultured at 30° C.)

In all the tested media, the hydrolysis of starch is observed and thegrade of hydrolysis is medium.

(4) Coagulation and peptonization of skimmed milk (skimmed milk,cultured at 37° C.)

From about the fifth day of the incubation, skimmed milk can becoagulated and immediately undergoes complete coagulation, followed bystarting of the peptonization. The progress of the peptonization isslow. The peptonization was not completed in three weeks of observation.

(5) Formation of melanoid pigments (tryptone-yeast broth, ISP-Medium 1;peptone-yeast-iron-agar medium, ISP-Medium 6; tyrosine-agar medium,ISP-Medium 7; all cultured at 30° C.)

Formation of melanoid pigment is negative on the tryptone-yeast brothand the tyrosine-agar medium but is probably positive on thepeptone-yeast-iron-agar medium.

(6) Utilization of carbon sources (Pridham-Gottlieb agar medium,ISP-Medium 9, cultured at 30° C.)

L-arabinose, D-xylose, D-glucose, D-fructose, rhamnose and D-mannitolare utilizable for growth, but sucrose, inositol, raffinose and lactoseare not utilizable.

(7) Liquefaction of calcium malate (calcium malate-agar medium, culturedat 30° C.)

Liquefaction of calcium malate is not observed.

(8) Reduction of nitrate (aqueous solution of peptone containing 0.1% ofpotassium nitrate, ISP-Medium 8, cultured at 30° C.)

The reduction was positive.

(9) Decomposition of cellulose (Synthetic test solution containingpieces of filter paper added, cultured at 30° C.)

The decomposition was negative.

Summarizing the above-mentioned characteristics, MI215-NF3 strain ismorphollogically characterized in that aerial hyphae bear spore chainsof hooked or spiral shape and each chain of spores contains 7-15 spores.The surface of spore is warty. Neither whirls nor sporangia areobserved. In addition, splitting of substrate mycelia is not observed onvarious culture media, white to gray white or pink to white coloredaerial hyphae are formed on a growth of a pale yellow to pale yellowbrown to grayish yellow brown in color. Soluble pigment is produced withslight yellowish tinge.

Formation of melanoid pigments is negative on tryptone-yeast broth andtyrosine-agar medium but positive on peptone-yeast-iron-agar medium. Thegrade of protein-decomposing activity is medium to weak, while the gradeof the activity to hydrolyze starch is medium. Further, the reduction ofnitrate is positive.

Furthermore, MI215-NF3 strain contains mesodiaminopimelic acid as acomponent of the cell and contains glucose, galactose, madurose andribose as the sugar components in the whole cell, and it shows Type IIIBfor the principal constituents of the cell wall according to theproposal by Lechevalier et al. described in the "International Journalof Systematic Bacteriology", 20, 435 (1970). On the other hand, itsphospholipids are of the PI type (i.e., do not contain phosphatidylethanolamine, phosphatidyl xethylethanolamine, phosphatidyl choline andunknown glycosamine-containing phospholipids but contain phosphatidylinositol and phosphatidyl inositolmannoside). The menaquinone iscomposed of MK-9(H₆), MK-9(H₈) and a small amount of MK-9(H₄).

In view of the foregoing, MI215-NF3 strain is considered to be anactinomycete strain belonging to the genus Actinomadura. Among the knownstrains of the genus Actinomadura, microbial species analogous toMI215-NF3 strain have been searched for. Actinomadura citrea [Literature1: "Antibiotiki", 17, 965-970 (1970); and Literature 2: "Systematic &Applied Microbiology", 6, 264-270 (1985)] can be mentioned as theanologous species in view of its shape and menaquinone composition.Thus, MI215-NF3 strain and Actinomadura citrea are very close to eachother in that they form the spore chains of a hooked or spiral shape,have warty surface of spores and produce soluble pigments of yellowcolor on various culture media and that the menaquinone compositions oftheir cells contain MK-9(H₆) and MK-9(H₈). However, there remain stillmany aspects that cannot be determined from the literatures. The presentinventors are planning to obtain the standard strain of Actinomaduracitrea and conduct a comparative experiment with it. In account of thesematters, MI215-NF3 strain is named "Actinomadura sp. MI215-NF3" at thepresent time. Incidentally, MI215-NF3 strain was deposited on Nov. 22,1988 in "Fermentation Research Institute", Agency of Industrial Scienceand Technology, Ministry of International Trade and Industry, located atTsukuba-shi, Ibaraki-ken, Japan, and has been stored there under "FERMP-10397". In addition, MI215-NF3 strain has been deposited under "FERMBP-2675" in terms of the Budapest Treaty.

Further, in a third aspect of the present invention, there is alsoprovided an agent for controlling or therapeutically treating fowlcoccidiosis, which contains as an active ingredient MI215-NF3 substanceof the formula (I) or a salt thereof.

BEST MODES OF WORKING THE INVENTION

In the process according to the second aspect of the present invention,the production of MI215-NF3 substance is carried out by the followingprocedures.

Thus, the production of MI215-NF3 substance may preferably be conductedby cultivation of an MI215-NF3 substance-producing microorganism in anutrient culture medium under submerged aerobic conditions at atemperature of 27-30 ° C. and under agitation. The nutrient mediumemployable for that purpose may contain an assimilable carbon sourcesuch as sugar, starch, glycerin or molasses, and an organic nitrogensource such as corn steep liquor, soybean flour, cotton seed flour,peptone, meat extract or hop cake, as well as an inorganic nitrogensource such as ammonium sulfate, ammonium nitrate or ammonium chloride.If excessive foaming occurs in the course of fermentation, a defoamingagent such as a vegetable oil or silicone may be added to thefermentation medium. If the pH of the medium during the fermentationvaries, a buffering agent such as calcium carbonate may be added. Tomake the aeration of the culture medium in a submerged incubation tank,it is preferable to continuously feed into the culture medium a flow offresh air at a rate of about 1/2 to 2 parts by volume per part by volumeof the culture medium broth and per minute. Stirring of the medium maybe effected using a stirring means well known in the industry offermentation.

As a seed culture for the production of MI215-NF3 substance, a culturedmaterial obtained by slant-cultivation of MI215-NF3 strain on an agarmedium is employed. This cultured material may be used for inoculationto the culture medium either in a flask for shaking incubation or in atank for preparation of seed culture. As an alternative, a growthproduct as obtained from such a shaking incubation flask can beinoculated to the seed-culturing tank.

MI215-NF3 strain usually can grow to a maximum in 5 to 7 days ofincubation. However, the time required until a microorganism employedreaches the maximum growth varies depending on the fermentatingapparatus, aeration rate, stirring speed and so on. In general, thefermentation is continued until sufficient anti-bacterial potency hasbeen imparted to the culturing medium. Time-dependent variations of thepotency of MI215-NF3 substance in the culture broth can be determined bythe cup plate assay method using Staphylococcus aureus Smith as a testmicroorganism.

In the process of the present invention, MI215-NF3 substance isrecovered from the culture as obtained as above. The recovery ofMI215-NF3 substance can be achieved suitably by using such measureswhich are generally employed for the recovery of metabolic productsproduced by a microorganism. MI215-NF3 substance can be recovered, forexample, by employing, either singly or in combination, such a methodwhich makes use of a difference in the solubility between the desiredproduct and an impurity, such a method which utilizes differences in theadsorption affinity to various adsorbents, and such a method in whichthe desired product is extracted with a water-immiscible solvent, andthe like. In principle, it is preferred to recover MI215-NF3 substanceby the following method. Thus, the microbial cells are separated fromthe culture by filtration or centrifugation. From the resultant filtrateof the culture, the MI215-NF3 substance is extracted with awater-immiscible organic solvent such as n-butyl acetate or ethylacetate. The resulting extract is then washed with water, and theorganic solvent is distilled off from the extract to dryness. Theresulting dry residue usually contains MI215-NF3 substance in aproportion of about 10%. To increase the purity of a crude MI215-NF3substance obtained as the dry residue, the dry residue may be subjectedto purification by chromatography with a silica gel column so thatMI215-NF3 substance of 95% purity or higher can be obtained. MI215-NF3substance obtained in the manner described above is usually in the formof a salt such as the sodium or potassium salt or a free carboxylicacid. It can be converted into a single salt by a method known per se inthe art. For example, the sodium salt of MI215-NF3 substance may beobtained as colorless plate crystals by dissolving the above MI215-NF3substance in ethyl acetate, washing the resultant organic solutionsuccessively with aqueous 1 mole HCl solution and a saturated aqueoussodium carbonate solution, drying the organic solvent phase overanhydrous sodium sulfate, concentrating the dried solution to drynessand then crystallizing the resultant solid from ethyl acetate-hexane.

MI215-NF3 substance in the free carboxylic acid form can be formed bydissolving MI215-NF3 substance (sodium salt), which has been obtained bypurifying a crude MI215-NF3 substance as described just above, in anorganic solvent such as ethyl acetate or methanol, and then treating theresultant solution with a strongly acidic ion-exchange resin (H⁺ form)added therein. When MI215-NF3 substance in the free acid form is treatedin an organic solvent, for example, with a basic alkali metal salt,e.g., an aqueous solution of sodium carbonate or potassium carbonate orwith a basic water-soluble alkaline earth metal compound, e.g., calciumhydroxide or magnesium hydroxide, MI215-NF3 substance in the form of thecorresponding alkali metal salt or alkaline earth metal salt can beproduced. Similarly, it can also be treated with another metal salt, forexample, aluminum hydroxide or ferric hydroxide to prepare the aluminumor iron salt, correspondingly.

In accordance with the third aspect of this invention, the agent forcontrolling chicken coccidiosis contains MI215-NF3 substance of theformula (I) or a salt thereof as an active ingredient. In this agent,MI215-NF3 substance or its salt as the active ingredient compound may bemixed with a veterinarilly acceptable excipient, for example, starch,cellulose powder, woodmeal or ethanol or with a feed material, similarlyto the known drugs for similar applications. The amount of MI215-NF3substance or its salt in a composition which is prepared by mixingMI215-NF3 substance with such an excipient may be at a concentration of50-100 ppm which is effective for the therapeutic treatment ofcoccidiosis. The agent of the present invention for controlling thecoccidiosis may also be in the form of a solution of MI215-NF3 substanceor its salt in a suitable organic solvent or may be in the form of aconcentrate or dispersion of MI215-NF3 substance or its salt suspendedin a water-containing organic solvent. Upon use, such a solution orsuspension may be added to a feed so that the concentration of theactive ingredient substance is in a range from 50 ppm to 100 ppm in thefeed.

In a further aspect of the present invention, there is also providedActinomadura sp. MI215-NF3 strain which is a useful novel microorganismcapable of producing the useful antibiotic substance of the formula (I)as described above.

The present invention will hereinafter be illustrated in more detailwith reference to Examples.

EXAMPLE 1

Cells of a slant culture of Actinomadura sp. MI215-NF3 strain (FERMBP-2675) were inoculated to a rotary flask of 500-ml capacity containing110 ml of a seed culture medium which comprised 2% galactose, 2% dextrinfor chemical use, 1% soy peptone, 0.5% corn steep liquor, 0.2% ammoniumsulfate and 0.2% calcium carbonate and had pH 7.4. The incubation wasmade at 30° C. for 144 hours on a rotating shaker. The culture broththus obtained was inoculated at a rate of 3 wt.% to 500-ml rotary flaskseach containing 110 ml-aliquoat of a primary culture medium, whichcomprised 1.5% glycerin, 1.5% soluble starch, 0.5% soybean flour, 1.5%fish meal and 0.2% calcium carbonate and had pH 7.4, with the totalvolume of the primary culture medium being 5 litres. Each of thethus-inoculated culture media was incubated at 27° C. for 144 hours on arotating shaker at 180 rpm. The microbial cells were separated byfiltration from the culture thus obtained. About 4 1 of the resultingculture broth filtrate were extracted with 3 1 of ethyl acetate, and theresulting extract was washed with water and then dried over anhydroussodium sulfate. The dried ethyl acetate solution was filtered to removeinsoluble matters, and the filtrate was evaporated to dryness. Theresidue was dissolved in 100 ml of ethyl acetate. Insoluble matters wereagain filtered off, and the filtrate was evaporated to dryness so that2.4 g of a crude product of MI215-NF3 substance were obtained.

EXAMPLE 2

In chloroform were dissolved 2.4 g of the crude product of MI215-NF3substance as obtained in Example 1. The resulting solution was subjectedto chromatography in a column of 150 g of "Xerogel 60" (trade name), asilica gel produced by Merck & Co., Inc., packed with aid of chloroform.The silica gel column was then eluted using chloroform-acetone (5:1) asa developer solvent. MI215-NF3 substance was eluted out in 1600 ml to3700 ml fractions of the eluate. After the solvent was distilled offfrom the active fractions under reduced pressure, the residue wasdissolved in 90 ml of ethyl acetate. The resultant solution wassuccessively washed with aqueous 0.1 M HCl solution, aqueous 0.1 Msodium hydroxide solution and saturated saline, followed by drying overanhydrous sodium sulfate. The dried solution was thereafter distilledunder reduced pressure to remove the solvent, whereby 255 mg of an oilysubstance was obtained. The oil obtained was crystallized from a mixedsolvent of hexane-ethyl acetate so that the sodium salt of MI215-NF3substance was afforded as colorless plate crystals in a yield of 232 mg.

EXAMPLE 3

In 100 ml of chloroform were dissolved 200 mg of the sodium salt ofMI215-NF3 substance. The solution was added with 10 ml of water and thenadjusted to pH 3.0 with hydrochloric acid. The resulting mixture wassubjected to shaking extraction so that a chloroform layer was obtained-After the chloroform layer as separated was washed with water, it wasdried over anhydrous sodium sulfate and then concentrated to dryness.MI215-NF3 substance in the free carboxylic acid form was obtained in ayield of 196 mg.

EXAMPLE 4

MI215-NF3 substance (20 mg) was dissolved in 1 ml of acetone, to which0.3 ml of a suspension of 2.5% of aluminum hydroxide was added. Afterreaction at room temperature for 18 hours, 100 ml of water were added tothe reaction solution, and the resultant mixture was extracted twicewith 100 ml of chloroform. The chloroform layer was washed with water,dried and then concentrated to dryness, to give 19.8 mg of the aluminumsalt of MI215-NF3 substance.

INDUSTRIAL UTILIZABILITY OF THE INVENTION

As has been described above, the novel antibiotic, MI215-NF3 substance,or a salt thereof can be obtained by culturing Actinomadura sp.MI215-NF3 strain which has the capability to produce the antibioticMI215-NF3 substance, and then isolating it from the culture. MI215-NF3substance and its salts have activities to control or therapeuticallytreat chicken coccidiosis and also useful antibacterial activitiesagainst certain species of bacteria. No particular limitation is imposedon the species of fowls whose infected coccidiosis can be treated byMI215-NF3 substance and its salts according to the present invention. Inaddition, the antibiotic MI215-NF3 substance and its salts according tothe present invention are more effective as compared with conventionalanticoccidial antibiotics such as monensin and salinomycin even at asmaller dosage.

The present invention can therefore provide an antibiotic, MI215-NF3substance and salts thereof which are useful as drugs for animals and asa medicine for men.

We claim:
 1. An antibiotic, MI215-NF3 substance, having the followingformula (I): ##STR2## wherein R is a hydrogen atom; and a salt ofMI215-NF3 substance of the formula (I) where R is a metal selected fromthe group, alkaline metal, alkaline earth metal, iron, aluminum andammonium ion.
 2. A composition for controlling chicken coccidiosis,comprising as an active ingredient MI215-NF3 substance having theformula (I) defined in claim 1, or a salt thereof as defined in claim 1and a carrier therefor.